Our laboratory studies mechanisms of phosphoinositide 3-kinase (PI3K) signaling in lymphocytes. We are currently funded by grant # 1 RO1-AI50831-01 to determine the specific roles of individual PI3K isoforms in lymphocyte activation. This work relies on standard functional and biochemical assays to characterize lymphocytes from mice lacking specific PI3K isoforms. However, traditional biochemical approaches to measuring signaling enzyme activity have significant limitations, especially when applied to primary lymphocytes. Advances in single cell analysis of signaling events offer powerful tools to advance our understanding of PI3K, as well as other critical signaling systems. In keeping with the developmental emphasis of this R21 mechanism, this application seeks to develop an emerging technology, the laser micropipet system (LMS), to study PI3K-dependent signals at the single cell level. Our first objective is to develop optimal conditions for studying lymphocytes using LMS. Our second aim is to apply this technology to address the role of the PI3K regulatory isoform p85alpha in activation of different downstream signaling molecules.